Showing papers by "Rakesh K. Jain published in 1996"

Time-Dependent Vascular Regression and Permeability Changes in Established Human Tumor Xenografts Induced by an Anti-Vascular Endothelial Growth Factor/Vascular Permeability Factor Antibody

Abstract: The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 micrograms/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/ VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression-i.e., blocking the interactions between VEFG/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.

During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium.

Abstract: Localization of activated natural killer (A-NK) cells in the microvasculature of growing tumors is the result of recognition of the intracellular and vascular cell-adhesion molecules ICAM-1 and VCAM-1 on the tumor endothelium, mediated by lymphocyte function-associated protein LFA-1 and vascular lymphocyte function-associated protein VLA-4. In vitro and in vivo studies of A-NK cell adhesion to endothelial cells showed that vascular endothelial growth factor (VEGF) promotes adhesion, whereas basic fibroblast growth factor (bFGF) inhibits adhesion through the regulation of these molecules on tumor vasculature. Thus, some angiogenic factors may facilitate lymphocyte recognition of angiogenic vessels, whereas others may provide such vessels with a mechanism that protects them from cytotoxic lymphocytes.

Delivery of Molecular Medicine to Solid Tumors

Abstract: Molecular medicine is increasing our understanding of the molecular basis of cancer and is providing new targets for therapeutic intervention But at present we do not have appropriate ways to deliver these agents to the tumor, and without this ability their potential will not be realized Jain proposes a three-part strategy for overcoming this obstacle to successful treatment of tumors

Quantitation and physiological characterization of angiogenic vessels in mice: effect of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and host microenvironment.

Abstract: A prerequisite for the development of novel angiogenic and anti-angiogenic agents is the availability of routine in vivo assays that permit 1) repeated, long-term quantitation of angiogenesis and 2) physiological characterization of angiogenic vessels. We report here the development of such an assay in mice. Using this assay, we tested the hypothesis that the physiological properties of angiogenic vessels governed by the microenvironment and vessel origin rather than the initial angiogenic stimulus. Gels containing basic fibroblast growth factor (bFGF) or vascular endothelial growth (VEGF) were implanted in transparent windows in the dorsal skin or cranium of mice. Vessels could be continuously and non-invasively monitored and easily quantified for more than 5 weeks after gel implantation. Newly formed vessels were first visible on day 4 in the cranial window and day 10 in the dorsal skinfold chamber, respectively. The number of vessels was dependent on the dose of bFGF and VEGF. At 3000 ng/ml, bFGF- and VEGF-induced blood vessels had similar diameters, red blood cell velocities, and microvascular permeability to albumin. However, red blood cell velocities and microvascular permeability to albumin were higher in the cranial window than in the dorsal skinfold chamber. Leukocyte-endothelial interaction was nearly zero in both sites. Thus, newly grown microvessels resembled vessels of granulation and neoplastic tissue in many aspects. Their physiological properties were mainly determined by the microenvironment, whereas the initial angiogenic response was stimulated by growth factors.

Tumor Angiogenesis and Interstitial Hypertension

Abstract: Due to the high permeability of tumor vessels to fluids and plasma proteins, the microvascular pressure (MVP) is the principal driving force for interstitial hypertension in solid tumors; as a result, hydrostatic pressures between the microvascular and interstitial space are close to equilibrium. Based on these observations, we hypothesized that the tumor interstitial fluid pressure (IFP) should increase following the onset of angiogenesis. To this end, the relationship between IFP and tumor neovascularization was determined in the human colon adenocarcinoma (LS174T) and the murine carcinoma (MCaIV) implanted in a transparent dorsal skin fold chamber in severe combined immunodeficient mice. Three stages in the development of the tumor neovasculature were characterized by intravital microscopy. Stage I tumors were avascular, stage II was characterized by vascular sprouts and loops, and in stage III, the tumor vasculature was completely developed and blood flow was obvious. The IFP was measured with micropipettes and a servo-null system. For both tumor types, the IFP in stage I tumors was close to 0 mm Hg, and IFP increased significantly from one stage to the next. To further confirm that interstitial hypertension was associated with the development of the tumor vasculature, IFP was measured in LS174T spheroids. The mean pressure in spheroids was 0.2 +/- 0.3 mm Hg. In stage III tumors, the IFP was compared to the MVP. In MCaIV, the MVP was comparable to the IFP; however, in LS174T the MVP was significantly higher than the IFP. In conclusion, the results demonstrate that avascular tumors have atmospheric pressures and that tumor interstitial hypertension is associated with the development of the neovasculature.

Compatibility and the genesis of residual stress by volumetric growth.

Abstract: The equations of compatibility which are pertinant for growth strain fields are collected and examples are given in simply-connected and multiply-connected regions. Compatibility conditions for infinitesimal strains are well known and the possibilities of Volterra dislocations in multiply-connected regions are enumerated. For finite growth strains in a multiply-connected regions, each case must be examined individually and no generalizations in terms of Volterra dislocations are available. Any incompatible growth strains give rise to residual stresses which are known to occur in many tissues such as the heart, arterial wall, and solid tumors.

Transport in lymphatic capillaries. I. Macroscopic measurements using residence time distribution theory

Abstract: We present a novel integrative method for characterizing transport in the lymphatic capillaries in the tail of the anesthetized mouse, which is both sensitive and reproducible for quantifying uptake and flow Interstitially injected, fluorescently labeled macromolecules were used to visualize and quantify these processes Residence time distribution (RTD) theory was employed to measure net flow velocity in the lymphatic network as well as to provide a relative measure of lymphatic uptake of macromolecules from the interstitium The effects of particle size and injection pressure were determined The uptake rate was found to be independent of particle size in the range of a 6- to 18-nm radius; beyond this size, the interstitial matrix seemed to pose a greater barrier A comparison of 10 vs 40 cmH2O injection pressure showed a significant influence on the relative uptake rate but not on the net velocity within the network (33 +/- 08 vs 38 +/- 10 micron/s) This suggested the presence of a systemic driving force for baseline lymph propulsion that is independent of the local pressure gradients driving the uptake This model can be used to examine various aspects of transport physiology of the initial lymphatics

Effect of Radiation on Interstitial Fluid Pressure and Oxygenation in a Human Tumor Xenograft

Abstract: Elevated interstitial fluid pressure (IFP) is a pathophysiological characteristic of most human and experimental tumors and may be responsible, in part, for the poor distribution of blood-borne therapeutic agents and low blood flow rate in tumors. Recent data in cervical carcinomas in patients suggest that fractionated radiation can lower tumor IFP and increase oxygen partial pressure (pO (2)) in some patients. The goals of this study were to find the minimum dose of radiation required to modulate IFP and pO(2) and to determine the time course of IFP changes due to radiation in a preclinical model. Xenografts of the LS174T human colon adenocarcinoma were grown in the right flank of nude (BALB/c) mice. IFP and pO(2) were measured before and 24 h after graded doses of irradiation. The mean +/- SD initial IFP in untreated tumors was 12.9 +/- 0.5 mm Hg (n=109), and the range was 3.0 to 40.3 mm Hg. The mean +/- SD and median initial pO(2) were 20.2 +/- 2.4 and 11.9 mm Hg, respectively (n=37). IFP and pO(2) were independent of tumor size. Fractionated radiation lowered IFP by 2.5 mm Hg when the total dose was 10 or 15 Gy (P 0.05). Irradiation increased the proportion of tumors at higher oxygen tensions when compared to control tumors. The IFP and tumor volumes were followed for up to 10 days after a single dose of 10, 20, or 30 Gy of irradiation. IFP decreased for all treatment groups. The decrease was most significant for the group receiving 30 Gy. On day five following irradiation, the IFP had decreased by 35%. The changes in IFP and pO(2) occurred before any macroscopic changes in tumor volume could be observed. The radiation-induced decrease in IFP could be, in part, responsible for the increased uptake of monoclonal antibodies following single or fractionated radiation that has been reported in the literature.

Reduction of interstitial fluid pressure after TNF-alpha treatment of three human melanoma xenografts.

Abstract: Tumour necrosis factor-alpha (TNF-alpha) reduced the interstitial fluid pressure (IFP) to 54-64% (P < 0.05) and the mean arterial blood pressure (MABP) to 70% (P < 0.01) of control values after 5 h in three human melanoma tumour lines transplanted to nude mice.

Leukocyte-endothelial adhesion and angiogenesis in tumors

Abstract: Leukocyte-endothelial adhesion and angiogenesis, until recently considered as separate processes, have been shown to be linked by two recent findings: soluble cellular adhesion molecules (CAMs) involved in leukocyte-endothelial interactions are angiogenic and well known angiogenic molecules secreted by cancer or immune. cells can modulate the endothelial CAMs. This molecular link may partially explain why the overall leukocyte-endothelial interaction is often low and heterogeneous in angiogenic tumor vessels and why activated lymphocytes adhere nonuniformly to tumor vessels when injected into the tumor's blood supply.

Physiologically based kinetic model of effector cell biodistribution in mammals: implications for adoptive immunotherapy.

Abstract: The goal of the present investigation was to develop a physiologically based kinetic model to describe the biodistribution of immunologically active effector cells in normal and neoplastic tissues of mammals based on the current understanding of lymphocyte trafficking pathways and signals. The model was used to extrapolate biodistribution among different animal species and to identify differences among different effector populations and between intra-arterial and systemic injections. Most importantly, the model was used to discern critical parameters for improving the delivery of effector cells. In the model, the mammalian body was divided into 12 organ compartments, interconnected in anatomic fashion. Each compartment was characterized by blood flow rate, organ volume and lymphatic flow rate, and other physiological and immunological parameters. The resulting set of 45 differential equations was solved numerically. The model was used to simulate the following biodistribution data: (a) nonactivated T lymphocytes in rats; (b) interleukin 2-activated tumor-infiltrating lymphocytes in humans; (c) nonactivated natural killer (NK) cells in rats; and (d) interleukin 2-activated adherent NK cells in mice. Comparisons between simulations and data demonstrated the feasibility of the model and the scaling scheme. The similarities as well as differences in biodistribution of different lymphocyte populations were revealed as results of their trafficking properties. The importance of lymphocyte infiltration from surrounding normal tissues into tumor tissue was found to depend on lymphocyte migration rate, tumor size, and host organ. The study confirmed that treatment with effector cells has not been as impressive as originally promised, due, in part, to the biodistribution problems. The model simulations demonstrated that low effector concentrations in the systemic circulation greatly limited their delivery to tumor. This was due to high retention in normal tissues, especially in the lung. Reducing normal tissue retention through decreasing attachment rate or adhesion site density in the lung by 50% could increase the tumor uptake by approximately 40% for tumor-infiltrating lymphocytes and by approximately 60% for adherent NK cells. Our analysis suggested the following strategies to improve effector cell delivery to tumor: (a) bypassing the initial lung entrapment with administration to the arterial supply of tumor; (b) reducing normal tissue retention using effector cells with high deformability or blocking lymphocyte adhesion to normal vessels; and (c) enhancing tumor-specific capture and arrest by modifying the tumor microenvironment.

Fluorescence ratio imaging of interstitial pH in solid tumours: effect of glucose on spatial and temporal gradients.

Abstract: Tumour pH plays a significant role in cancer treatment. However, because of the limitations of the current measurement techniques, spatially and temporally resolved pH data, obtained non-invasively in solid tumours, are not available. Fluorescence ratio imaging microscopy (FRIM) has been used previously for noninvasive, dynamic evaluation of pH in neoplastic tissue in vivo (Martin GR, Jain RK 1994, Cancer Res., 54, 5670-5674). However, owing to problems associated with quantitative fluorescence in thick biological tissues, these studies were limited to thin (50 microns) tumours. We, therefore, adapted the FRIM technique for pH determination in thick (approximately 2 mm) solid tumours in vivo using a pinhole illumination-optical sectioning (PIOS) method. Results show that (1) steep interstitial pH gradients (5 microns resolution), with different spatial patterns, exist between tumour blood vessels; (2) pH decreased by an average of 0.10 pH units over a distance of 40 microns away from the blood vessel wall, and by 0.33 pH units over a 70 microns distance; (3) the maximum pH drop, defined as the pH difference between the intervessel midpoint and the vessel wall, was positively correlated with the intervessel distance; (4) 45 min following a systemic glucose injection (6 g kg-1 i.v), interstitial pH gradients were shifted to lower pH values by an average of 0.15 pH units, while the spatial gradient (slope) was maintained, when compared with preglucose values. This pH decrease was not accompanied by significant changes in local blood flow. pH gradients returned to near-baseline values 90 min after glucose injection; (5) interstitial tumour pH before hyperglycaemia and the glucose-induced pH drop strongly depended on the local vessel density; and (6) sodium bicarbonate treatment, either acute (1 M, 0.119 ml h-1 for 3 h i.v.) or chronic (1% in drinking water for 8 days), did not significantly change interstitial tumour pH. Modified FRIM may be combined with other optical methods (e.g. phosphorescence quenching) to evaluate non-invasively the spatial and temporal characteristics of extracellular pH, intracellular pH and pO2 in solid tumours. This will offer unique information about tumour metabolism and its modification by treatment modalities used in different cancer therapies.

Transport in lymphatic capillaries. II. Microscopic velocity measurement with fluorescence photobleaching

Abstract: Despite its relevance to the physiology of lymph formation and propulsion, the instantaneous flow velocity in single lymphatic capillaries has not been measured to date The method of fluorescence recovery after photobleaching (FRAP) was adapted for this purpose and used to characterize flow in the lymphatic capillaries in tail skin of anesthetized mice during a constant-pressure intradermal injection of fluorescein isothiocyanate-dextran (mol wt 2 x 10(6) The median lymph flow velocity was 47 microns/s, and the velocity magnitude ranged from 0 to 29 microns/s The direction of flow was generally proximal, but stasis and backflow toward the site of injection was also detected Evidence for oscillatory flow was detected in some FRAP experiments, and in separate experiments a periodicity of approximately 120 min-1, directly correlated to respiration frequency, was measured by tracking the motion of fluorescent latex microspheres (1 micron diam) introduced into the lymphatic capillary network The velocity magnitude showed a correlation with duration of infusion but not with distance from injection site It is speculated that the temporal decay of mean velocity magnitude could be related to the relaxation of local pressure gradients as partially collapsed vessels expand during the infusion

1995 Whitaker Lecture: delivery of molecules, particles, and cells to solid tumors.

Abstract: To reach cancer cells in a tumor, a blood-borne therapeutic agent must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium, and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically, and then to integrate the resulting information in a unified theoretical framework. This paradigm of analysis and synthesis has allowed us to obtain a better understanding of physiological barriers in solid tumors, and to develop novel strategies to exploit and/or to overcome these barriers for improved cancer detection and treatment.

Perfusion of single tumor microvessels: application to vascular permeability measurement.

Abstract: Objective: To develop a new method for determining the relative importance of convection versus diffusion in macromolecular transport across tumor microvessel walls.Methods: The human colon adenocarcinoma LS174T was transplanted in the dorsal skinfold chamber in a severe combined immunodeficient (SCID) mouse. The vasculature at the tumor surface was exposed by carefully removing the glass window of the chamber. A tumor microvessel was randomly selected, which was ∼20–40 μm in diameter, embedded in the connective tissue 10–12 μm below the surface of the tumor. The vessel was cannulated with a micropipette and perfused with fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) at different perfusion pressures. The fluorescence intensity was recorded on videotapes via a video system attached to the fluorescence microscope for offline analysis. The apparent vascular permeability was determined based on the time-dependence of fluorescence intensity and the vessel diameter.Results: The apparent v...

Pharmacokinetic analysis of the microscopic distribution of enzyme-conjugated antibodies and prodrugs: comparison with experimental data.

Abstract: A mathematical model was developed to improve understanding of the biodistribution and microscopic profiles of drugs and prodrugs in a system using enzyme-conjugated antibodies as part of a two-step method for cancer treatment. The use of monoclonal antibodies alone may lead to heterogeneous uptake within the tumour tissue; the use of a second, low molecular weight agent may provide greater penetration into tumour tissue. This mathematical model was used to describe concentration profiles surrounding individual blood vessels within a tumour. From these profiles the area under the curve and specificity ratios were determined. By integrating these results spatially, average tissue concentrations were determined and compared with experimental results from three different systems in the literature; two using murine antibodies and one using humanised fusion proteins. The maximum enzyme conversion rate (Vmax) and the residual antibody concentration in the plasma and normal tissue were seen to be key determinants of drug concentration and drug-prodrug ratios in the tumour and other organs. Thus, longer time delays between the two injections, clearing the antibody from the blood stream and the use of 'weaker' enzymes (lower Vmax) will be important factors in improving this prodrug approach. Of these, the model found the effective clearance of the antibody outside of the tumour to be the most effective. The use of enzyme-conjugated antibodies may offer the following advantages over the bifunctional antibody-hapten system: (i) more uniform distribution of the active agent; (ii) higher concentrations possible for the active agent; and (iii) greater specificity (therapeutic index).

Specificity analysis of three clonal and five non-clonal alpha 1,3-L-fucosyltransferases with sulfated, sialylated, or fucosylated synthetic carbohydrates as acceptors in relation to the assembly of 3'-sialyl-6'-sulfo Lewis x (the L-selectin ligand) and related complex structures.

Abstract: Unique specificities of the cloned α1,3-l-fucosyltransferases (FTs), FT III (Lewis type), FT IV (myeloid type), and FT V (plasma type), and the α1,3-FTs of Colo 205 (colon carcinoma), HL 60 (myeloi...

Oxygenation in tumors by modified hemoglobins.

Abstract: The effect of systemic injection of modified hemoglobin (Hb) prepared from bovine, human, or mouse Hb on tumor oxygenation was investigated. Hb was modified by (1) diisothiocyanatobenzenesulfonate (DIBS) to yield cross-linking within a tetramer; (2) glycolaldehyde (Glyal) to yield cross-linking between and within tetramers; (3) carboxymethylation (Cm) to change oxygen affinity; or (4) poly(ethylene glycol) (PEG) to yield attachment between tetramers. HGL9 (human glioma) in nude mice and FSaII (mice fibrosarcoma) in C3H mice were used as tumor models. Dose and time dependency were detected in the oxygenation effect by bovine-PEG-Hb. Internal cross-linkage prolonged the half-life in the circulation, and thus showed a significant effect. Compared to bovine-CmHb, bovine-DIBS-Hb and bovine-DIBS-CmHb were more effective. Decreasing the oxygen affinity by Cm significantly enhanced tumor oxygenation. Human-DIBS-CmHb was more effective than human-DIBS-Hb. These effects were caused by oxygen carrying capacity of modified Hbs as well as hemodynamic factors, and the injection seemed to reduce both perfusion-limited (acute) and diffusion-limited (chronic) hypoxia.

Intratumor pharmacokinetics, flow resistance, and metabolism during gemcitabine infusion in ex vivo perfused human small cell lung cancer.

Abstract: The relationship between tumor physiology and the pharmacokinetics of 2',2' difluorodeoxycytidine [gemcitabine (dFdC)] in ex vivo perfused human small cell lung cancer was examined. Two small cell lung cancer sublines, 54A and 54B, with known in vivo sensitivity to dFdC, were grown as tissue-isolated tumors in athymic mice and perfused ex vivo with or without 20-40 micrometer dFdC. Arteriovenous differences in gases, pH, and metabolites were determined before and during drug infusion. The geometric flow resistance (FR) of individual tumors was calculated, and dFdC and its inactive metabolite 2',2' difluorodeoxyuridine were determined by high-performance liquid chromatography of consecutive samples from the output line. Both tumors had prominent lactate production concurrent with a significant O2 consumption. The arteriovenous pH drop was approximately 0.3 in both tumor lines. Significant metabolic differences between 54A and 54B tumors were found that elucidated previously described differences further. Pharmacokinetic analysis showed that the initial tumor uptake of dFdC was flow limited, and a significant inverse correlation between the geometric FR and initial drug uptake was found. The rate constant for recovery of the drug in the tumor outflow was greater in 54B tumors (P < 0.05), and the geometric FR was greater in 54A tumors (P < 0.01). The drug conversion rate was independent of physiological parameters. Attempts to modify the delivery of dFdC should be directed at the tumor blood flow distribution. More generally, our experimental model provides useful new insight into metabolism and intratumor pharmacokinetics of chemotherapeutic agents in solid tumors.

Method of enhancing delivery of a pharmaceutical formulation

Abstract: The invention provides a method of enhancing the amount of a pharmaceutical composition delivered to a target tissue site in a mammal, by creating a transient differential between tile hydrostatic pressure in the target site and a region near the target tissue site whereby the composition is transported toward the site. An apparatus for performing the method is provided. In one form that apparatus includes a pharmaceutical reservoir, pump, and an agent reservoir and pump.